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Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS resulting from a progressive loss of oligodendrocytes. Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes of the brain, and postmortem sections show concurrent loss of myelin basic protein and TAL from sites of demyelination. Infiltrating CD8+ CTLs are thought to play a key role in oligodendrocyte cell death. Cleavage by granzyme B (GrB) is predictive for autoantigenicity of self-proteins, thereby further implicating CTL-induced death in the initiation and propagation of autoimmunity. The precursor frequency and CTL activity of HLA-A2–restricted TAL 168–176–specific CD8+ T cells is increased in MS patients. In this paper, we show that TAL, but not myelin basic protein, is specifically cleaved by human GrB. The recognition site of GrB that resulted in the cleavage of a dominant TAL fragment was mapped to a VVAD motif at aa residue 27 by N-terminal sequencing and confirmed by site-directed mutagenesis. The major C-terminal GrB cleavage product, residues 28–337, had no enzymatic activity but retained the antigenicity of full-length TAL, effectively stimulating the proliferation and CTL activity of PBMCs and of CD8+ T cell lines from patients with MS. Sera of MS patients exhibited similar binding affinity to wild-type and GrB-cleaved TAL. Because GrB mediates the killing of target cells and cleavage by GrB is predictive of autoantigen status of self proteins, GrB-cleaved TAL-specific T cell-mediated cytotoxicity may contribute to the progressive destruction of oligodendrocytes in patients with MS.

T cell receptors (TCR) adopt a similar orientation when binding with major histocompatibility complex (MHC) molecules, yet the biological mechanism that generates this similar TCR orientation remains obscure. We show here the cocrystallographic structure of a mouse TCR bound to a human MHC molecule not seen by the TCR during thymic development. The orientation of this xenoreactive murine TCR atop human MHC deviates from the typical orientation more than any previously determined TCR/MHC structure. This unique orientation is solely due to the placement of the TCR Vα domain on the MHC. In light of new information provided by this structure, we have reanalyzed the existing TCR/MHC cocrystal structures and discovered unique features of TCR Vα domain position on class I MHC that correlate with CD8 dependence. Finally, we propose that the orientation seen in TCR recognition of MHC is a consequence of selection during T cell development.

Both TCRs and Ab molecules are capable of MHC-restricted recognition of peptide/MHC complexes. However, such MHC restriction is the predominant mode of recognition by T cells, but is extremely rare for B cells. The present study asks whether the dichotomy in Ag recognition modes of T and B cells could be due to fundamental differences in the methods by which TCRs and Abs recognize peptide/MHC complexes. We have compared MHC and peptide recognition by panels of CTL lines specific for the Tax and M1 peptides presented by HLA-A2 plus Tax and M1 peptide/HLA-A2-specific human Fabs that were selected from a naive phage display library. Collectively, the results indicate both striking similarities and important differences between Fab and TCR recognition of MHC and peptide components of the Tax and M1/HLA-A2 complexes. These findings suggest that these two classes of immunoreceptors have solved the problem of specific recognition of peptide/MHC complexes by nonidentical mechanisms. This conclusion is important in part because it indicates that Ab engineering approaches could produce second-generation Ab molecules that more closely mimic TCR fine specificity. Such efforts may produce more efficacious diagnostic and therapeutic agents.

Functional discrimination between structurally similar self and foreign antigens is a main attribute of adaptive immunity. Here we describe two feedback mechanisms in T lymphocytes that together sharpen and amplify initial signaling differences related to the quality of T cell receptor (TCR) engagement. Weakly binding ligands predominantly trigger a negative feedback loop leading to rapid recruitment of the tyrosine phosphatase SHP-1, followed by receptor desensitization through inactivation of Lck kinase. In contrast, strongly binding ligands efficiently activate a positive feedback circuit involving Lck modification by ERK, preventing SHP-1 recruitment and allowing the long-lasting signaling necessary for gene activation. The characteristics of these pathways suggest that they constitute an important part of the mechanism allowing T cells to discriminate between self and foreign ligands.

The interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element. We provide a quantitative description of such interactions, which allows the identification of T cell epitopes and molecular mimics. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries composed of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. We demonstrate the predictive power of the novel strategy and show that, together with gene expression profiling by cDNA microarrays, it leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.

Binding of the TCR to class I or class II MHC molecules ever, a poorer correlation was observed between activity complexed with peptide is necessary for T cell activation and dissociation rate, as the TCR/peptide/MHC comand initiation of a cell-mediated immune response. Sim- plex with one of the antagonist peptides had a half-life ple alterations to antigenic peptides can result in a range almost twice as long as that observed with the weak of effects from loss of TCR signaling to weak or partial agonist. This prompted the authors to suggest that corsignaling or to the phenomenon of TCR antagonism relations observed between activity and dissociation (Sloan-Lancaster and Allen, 1996). Antagonist peptides rate may be coincidental, reflecting an underlying corredo not normally produce any of the outcomes associ- lation with affinity. ated with activation but result in an inhibition of the T X-ray crystallographic structures of abTCR/peptide/ cell response when the cell is subsequently challenged MHC complexes suggest that the entire range of TCRwith an activating agonist peptide.

The crystal structures of two human TCRs specific for a HTLV-I Tax peptide bound to HLA-A2 were recently determined, for the first time allowing a functional comparison of TCRs for which the MHC/peptide/TCR structures are known. Extensive amino acid substitutions show that the native Tax residues are optimal at each peptide position. A prominent feature of the TCR contact surface is a deep pocket that accommodates a tyrosine at position 5 of the peptide. For one of these TCRs, this pocket is highly specific for aromatic residues. In the other TCR structure, this pocket is larger, allowing many different residues to be accommodated. The CTL clones also show major differences in the specificity for several other peptide residues, including side chains that are not directly contacted by the TCR. Despite the specificity of these clones, peptides that are distinct at five or six positions from Tax11–19 induce CTL activity, indicating that substantial changes of the peptide surface are tolerated. Human peptides with limited sequence homology to Tax11–19 represent partial TCR agonists for these CTL clones. The distinct functional properties of these CTL clones highlight structural features that determine TCR specificity and cross-reactivity for MHC-bound peptides.

The purpose of TCR ligand binding is to initiate signal transduction that results in the activation of functional programs within the cell. How does binding of ligand on the extracellular V domains of the TCR translate into a functional message inside the cell? One model to explain these events is that ligand binding by the TCR V domains induces a conformational change in the C domains that, in turn, changes the dynamics of the interactions with CD3 chains and/or ζ chains associated with the receptor and thus transduces a signal through these associated components. However, a comparison of the liganded and unliganded forms of 2C showed that the conformational changes that occurred in the TCR as a result of antigen binding did not appear to involve the constant domains of the molecule (Garcia et al. 1998xStructural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Garcia, K.C, Degano, M, Pease, L.R, Huang, M, Peterson, P.A, Teyton, L, and Wilson, I.A. Science. 1998; 279: 1166–1172CrossRef | PubMed | Scopus (517)See all ReferencesGarcia et al. 1998). In addition, comparison of the Cα-Cβ interfaces of the liganded B7 TCR with unliganded 2C and N15 showed that they were nearly identical (Ding et al. 1998xTwo human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids. Ding, Y.-H, Smith, K.J, Garboczi, D.N, Utz, U, Biddison, W.E, and Wiley, D.C. Immunity. 1998; 8: 403–411Abstract | Full Text | Full Text PDF | PubMed | Scopus (359)See all ReferencesDing et al. 1998). Thus, the current structural evidence does not support the conformational change model.An alternate model for signal transduction by the TCR postulates that ligand binding induces receptor aggregation and/or oligomerization that segregates the bound receptors into cell–cell attachment patches (Davis et al. 1998xLigand recognition by αβ T cell receptors. Davis, M.M, Boniface, J.J, Reich, Z, Lyons, D, Hampl, J, Arden, B, and Chien, Y.-H. Annu. Rev. Immunol. 1998; 16: 523–544CrossRef | PubMed | Scopus (632)See all ReferencesDavis et al. 1998). Such receptor aggregation would facilitate clustering of intracellular components required for the recruitment and phosphorylation of signal transduction components. The adoption of a uniform orientation in TCR binding to peptide/MHC complexes may provide a mechanism that facilitates TCR aggregation and interactions with the coreceptors (CD3s, CD4/CD8) that can generate a T cell signal (Davis et al. 1998xLigand recognition by αβ T cell receptors. Davis, M.M, Boniface, J.J, Reich, Z, Lyons, D, Hampl, J, Arden, B, and Chien, Y.-H. Annu. Rev. Immunol. 1998; 16: 523–544CrossRef | PubMed | Scopus (632)See all ReferencesDavis et al. 1998). At this juncture, no evidence for dimerization or oligomerization of liganded TCRs is provided from the crystallographic data. However, the conditions used to produce these complexes may not favor multimerization, whereas the conditions of cell surface TCRs associated with CD3s/ζ chains could be much more favorable. Future structural studies of unliganded and liganded TCRs in association with the CD3 chains and ζ chains could provide new insights into the mechanisms of the initiation of signal transduction by the TCR.‡E-mail: web@helix.nih.gov and dgarboczi@atlas.niaid.nih.gov.

Human autoimmune diseases are thought to develop through a complex combination of genetic and environmental factors. Genome-wide linkage searches of autoimmune and inflammatory/immune disorders have identified a large number of non-major histocompatibility complex loci that collectively contribute to disease susceptibility. A comparison was made of the linkage results from 23 published autoimmune or immune-mediated disease genome-wide scans. Human diseases included multiple sclerosis, Crohn’s disease, familial psoriasis, asthma, and type-I diabetes (IDDM). Experimental animal disease studies included murine experimental autoimmune encephalomyelitis, rat inflammatory arthritis, rat and murine IDDM, histamine sensitization, immunity to exogenous antigens, and murine lupus (systemic lupus erythematosus; SLE). A majority (≈65%) of the human positive linkages map nonrandomly into 18 distinct clusters. Overlapping of susceptibility loci occurs between different human immune diseases and by comparing conserved regions with experimental autoimmune/immune disease models. This nonrandom clustering supports a hypothesis that, in some cases, clinically distinct autoimmune diseases may be controlled by a common set of susceptibility genes.

The demyelination process that occurs in the central nervous system (CNS) of patients with multiple sclerosis (MS) is due, in part, to an inflammatory response in which CD4+ and CD8+ T cells and macrophages infiltrate white matter. While it is thought that the inflammatory and demyelination process in MS is the product of Th1-associated cytokines secreted by CD4+ myelin protein-specific T cells present in the CNS, the mechanisms that are responsible for the recruitment and maintenance of these myelin-reactive CD4+ T cells in the CNS have not been elucidated. We have shown previously that CD8+CTL that recognize peptides derived from sequences of the myelin proteolipid protein (PLP) presented by HLA class I molecules can be generated in vitro, and that these PLP-specific CD8+CTL secrete the proinflammatory chemokines macrophage-inflammatory protein-1α and -1β, IL-16, and IP-10. In this study, we demonstrate that soluble products of these PLP-specific CD8+CTL can chemoattract CD4+ T cells that are specific for a myelin basic protein peptide and a PLP peptide, and that the majority of this chemotactic activity is mediated by IFN-inducible protein 10. These results demonstrate that PLP-specific CD8+ T cells can play a role in the recruitment and retention of myelin-derived peptide-specific CD4+ T cells, and indicate that they may play a proinflammatory role in the pathogenesis of MS.

Down-regulation of the initial burst of viremia during primary human immunodeficiency virus (HIV) infection is thought to be mediated predominantly by HIV-specific CD8+ cytotoxic T lymphocytes (CTL). This response is associated with major perturbations in the T cell receptor (TCR) repertoire. To investigate the failure of the cellular immune response to adequately control viral spread and replication and to prevent establishment of HIV infection, changes in the TCR repertoire and in the distribution of virus-specific CTL between blood and lymph node were analyzed in three patients with primary infection. By the combined use of clonotype-specific polymerase chain reaction and analysis of the frequency of in vivo activated HIV-specific CTL, it was shown that HIV-specific CTL clones preferentially accumulated in blood as opposed to lymph node. Accumulation of HIV-specific CTL in blood occurred prior to effective down-regulation of virus replication in both blood and lymph node. These findings should provide new insights into how HIV, and possibly other viruses, elude the immune response of the host during primary infection.

The demyelination process that occurs in the central nervous system (CNS) of patients with multiple sclerosis (MS) is in part due to an inflammatory response in which CD4+ and CD8+ T cells and macrophages infiltrate white matter. In this study, we have identified a peptide sequence derived from the CNS-specific myelin protein proteolipid protein (PLP) which could bind to HLA-A3 and induce a HLA-A3-restricted CD8+ CTL response from HLA-A3+ donors. These PLP peptide-specific CTL could lyse HLA-A3+ target cells pulsed with a homologous peptide derived from the CRM1 protein of Saccharomyces cerevisae. These findings demonstrate the immunogenic potential of a PLP-derived peptide for generation of autoreactive HLA-A3-restricted CD8+ CTL, and further show that these CTL can be activated by a peptide derived from a common environmental microorganism.

Most neural cells constitutively lack major histocompatibility complex (MHC) class I and beta 2-microglobulin gene expression. Cytokines and viruses may, however, induce expression of these genes in some neural cells, and this correlates with factor binding to the NF-kappa B and interferon stimulated response elements of these genes. Here, we demonstrate that NF-kappa B is capable of inducing MHC class I and beta 2-microglobulin gene expression when transiently co-transfected into CHP-126 neuroblastomas, and that IRF-2 represses this induction. Interferon regulatory factor-2 (IRF-2) repression of MHC class I and beta 2-microglobulin gene expression in CHP-126 neuroblastomas may demonstrate a mechanism by which virus persists in neural cells. We show here that IRF-2 physically interacts in vitro with NF-kappa B. This interaction may contribute to the repression of the expression of these genes. Our demonstration that IRF family members, in addition to IRF-2, physically interact in vitro with NF-kappa B (p50 and p65), provides a general mechanism by which these transcription factors may, in concert, regulate the expression of a variety of genes involved in immune responses in the brain.

Identification of the targets of autoreactive T cells is important for understanding the pathogenesis of many autoimmune diseases. In multiple sclerosis, myelin proteins are thought to be the targets of autoreactive T-cell responses. To date only major histocompatibility complex class II-restricted CD4+ T-cell responses to myelin proteins have been investigated. In the present study, the ability of self peptides derived from human myelin proteins to induce autoreactive CD8+ T-cell responses has been assessed. Peptide sequences from human myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and myelin oligodendrocyte glycoprotein have been identified that bind to and form stable complexes with HLA-A2. MBP 110-118, PLP 80-88, MAG 287-295, MAG 509-517, and MAG 556-564 were all able to induce peptide-specific HLA-A2-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses in vitro in HLA-A2+ individuals. CTLs specific for MBP 110-118 and MAG 556-564 could recognize endogenously processed antigens presented by HLA-A2. CTL clones reactive to MBP 110-118 and MAG 556-564 produced tumor necrosis factor alpha and a subset of these clones also produced interferon gamma. These results demonstrate that (i) self peptides derived from human myelin proteins can induce autoreactive CD8+ CTLs and (ii) these CD8+ T cells produce cytokines thought to be important in mediating demyelinating disease. These studies provide an experimental approach for the assessment of CD8+ T-cell responses in such autoimmune diseases.

An alternative pathway for class I-restricted antigen presentation has been suggested on the basis of peptides bound to HLA-A2 molecules in cells lacking the transporter for antigen presentation (TAP). Most of these peptides were derived from signal sequences for translocation into the endoplasmic reticulum (ER). However, it is not known whether these peptides can be presented to T cells. The hydrophobic nature of an HLA-A2-restricted T cell epitope (M1 58-66) was exploited to test whether it could be presented to T cells when derived from a signal sequence. Replacing the signal sequence of the influenza virus hemagglutinin molecule H3 with an artificial sequence containing that HLA-A2-restricted T cell epitope resulted in efficient translocation of H3 molecules into the ER and transport to the cell surface. This signal sequence-derived epitope was presented to HLA-A2-restricted T cells. Involvement of cytosolic processing for this presentation is very unlikely, because (a) presentation occurred in cells lacking TAP; (b) expression of H3 molecules with the artificial signal sequence did not produce a detectable cytosolic form of H3; and (c) presentation of the same epitope expressed in cytosolic forms of antigen required TAP. Thus, a peptide derived from a signal sequence cleaved in the ER can provide an epitope for HLA-A2-restricted T cell recognition.

The pathogenesis of multiple sclerosis (MS) is thought to involve a T-cell-mediated autoimmune process. Experimental allergic encephalomyelitis (EAE), an animal model resembling MS, can be induced by immunization with myelin antigens such as myelin basic protein. The T-cell antigen receptor (TCR) usage in EAE is highly restricted in some strains of animals and experimental treatments targeting the TCR have been successful in EAE. Examination of the TCR beta-chain variable-region (V beta) usage of MBP-specific T-cell lines in MS patients has produced conflicting results. Our previous studies of TCR alpha-chain variable-region usage in monozygotic twins demonstrated a general skewing of the TCR repertoire in individuals with MS. This skewing became apparent only after stimulation with antigens; in peripheral blood lymphocyte preparations from individuals with MS V alpha 8-bearing T cells were preferentially selected by stimulation with myelin basic protein. In the present study we examined complementarity-determining region 3 of those V alpha 8-positive TCRs. Marked sequence heterogeneity was found in all individuals with severe MS. In contrast, restricted areas of complementarity-determining region 3 were found in healthy control individuals and in individuals with a mild form of MS. Sequences from tetanus toxoid-specific V alpha 8-positive T cells generated from the same individuals were relatively homogeneous within individuals regardless of disease activity and were distinct from the sequences of complementarity-determining region 3 in myelin basic protein-stimulated lines. These findings suggest that disease severity may be associated with increased heterogeneity of myelin antigen-specific T cells and could reflect an impaired ability of the immune system to down-regulate these anti-self responses.

CARE-LASS is a highly sensitive, fast, simple and safe fluorometric microassay. Target cells are loaded with acetoxymethyl ester of calcein (calcein-AM) that passively crosses the cell membrane. Intracellular esterases convert the molecule to calcein, a polar fluorochrome which, in cells with intact plasma membranes, displays good retention characteristics and low pH sensitivity. In analogy to standard 51Cr release assays, the CARE-LASS system is based on the release of a marker into the supernatant that is measured by an automated fluorescence scanner and correlates with the number of lysed cells. We tested the CARE-LASS system by measuring cytotoxicity in major histocompatibility complex (MHC) class I and MHC class II restricted cytotoxic T lymphocyte (CTL) assays as well as lymphokine activated killer (LAK) mediated cytotoxicity. We applied a small set of target cell lines at various effector to target (E:T) ratios, at different antigen concentrations and compared CARE-LASS CTL data to data resulting from conventional 51Cr release assays. The CARE-LASS system provides a reliable and sensitive method to measure cell-mediated cytotoxicity.

Experiments were carried out to determine whether complexes between MHC class I molecules and synthetic peptides are representative of those formed under more physiologically relevant conditions, with peptides derived intracellularly from processed antigens. Lysis of cells sensitized with exogenously provided and endogenously generated peptide analogues of the optimal nonameric peptide 58–66 (GILGFVFTL; derived from the influenza virus matrix protein) was compared. Endogenous loading was accomplished by expressing minigene DNA coding for alanine-substituted analogues of peptide 58–66 in HLA-A2-positive cells. Susceptibility to lysis by HLA-A2-restricted, peptide-specific cytotoxic lymphocytes was compared with lysis of cells sensitized with the same synthetic peptides. Although results were quite comparable, differences were observed. The endogenously presented analogues 58–66L60A, G61A, T65A, and L66A were recognized more efficiently than the corresponding exogenously presented analogues. This difference in recognition was most striking for peptide 58–66G61A. These results indicate the need for caution in using synthetic peptides in defining peptide binding motifs. Additional experiments with endogenously expressed analogues of 58–66 with substitutions other than alanine were carried out to define the interaction between this peptide and HLA-A2. Results are compatible with the interpretation that residues 58, 59, and 60 interact with pockets A, B, and D, respectively, in the HLA-A2 binding groove and that these interactions contribute to peptide binding.

Although the cause of multiple sclerosis (MS) is unknown, it is thought to involve a T cell-mediated autoimmune mechanism. Susceptibility to the disease is influenced by genetic factors such as genes of the HLA and T-cell receptor (TCR) complex. Other evidence for a genetic influence includes the low incidence in certain ethnic groups, the increased risk if there are affected family members and the increased concordance rate for disease in monozygotic twin pairs (26%), compared to dizygotic twins. Epidemiological studies indicate that there may be an additional role for environmental factors. Although the target antigen(s) are not yet identified, several myelin or myelin-associated proteins have been suspected, among them myelin basic protein. A lack of genetically comparable controls has impaired the analysis of the T-cell response in MS patients and caused disagreement on TCR usage in the disease. Here we analyse the role of TCR genes in MS by comparing TCR usage in discordant versus concordant monozygotic twins in response to self and foreign antigens. We find that after stimulation with myelin basic protein or tetanus toxoid, control twin sets as well as concordant twin sets select similar V alpha chains. Only the discordant twin sets select different TCRs after stimulation with antigens. Thus exogenous factors or the disease shape the TCR repertoire in MS patients, as seen by comparison with unaffected genetically identical individuals. This skewing of the TCR repertoire could contribute to the pathogenesis of MS and other T-cell-mediated diseases.

To understand better the specificity of peptide binding by MHC class I molecules, we have evaluated the capacity of a panel of unrelated peptides to compete for the presentation of viral peptides presented by HLA-A3 and HLA-B27. The HIV-Nef7F peptide (74-82) was presented by HLA-A3 to Nef-specific HLA-A3-restricted CTL lines, and the influenza nucleoprotein peptide NP(380-393) was presented by HLA-B27 to NP(380-393)-specific HLA-B27-restricted CTL lines. In addition, we have extended studies from our group that have evaluated the capacity of a similar panel of peptides to inhibit presentation of an influenza nucleoprotein peptide NP (335-349) by HLA-B37 and a matrix peptide, M1 (57-68), by HLA-A2 to the appropriate peptide-specific CTL lines. Out of 41 peptides tested, only five bound to more than one of the MHC molecules analyzed. Pairwise comparisons of the peptide binding specificities among these four different class I molecules revealed no common competitor peptides in four of the six possible comparisons. Thus, each class I molecule appears to have a functionally distinct peptide binding site, as reflected by the ability to bind largely non-overlapping sets of peptides.

The ability of amino acid side chains in the floor of the peptide binding groove of HLA-A2 to affect the presentation of a viral peptide to peptide-specific cytotoxic T lymphocytes (CTL) has been examined. HLA-A2 molecules with naturally occurring single amino acid substitutions of Phe to Tyr at position 9 (HLA-A2.4a, Tyr9) and Tyr to Cys at position 99 (HLA-A2.4b, Cys99) and a site directed mutant with a Val to Leu substitution at position 95 (Leu95) were examined for their ability to present the influenza virus matrix M1 55-73 peptide and several sequence variants of the M1 peptide to a panel of 36 M1 55-73-specific HLA-A2.1-restricted CTL lines. The Leu95 molecule demonstrated enhanced kinetics of M1 peptide presentation and the ability to be sensitized by lower concentrations of the M1 peptide than the A2.1 molecule. The Tyr9 and Cys99 molecules exposed to M1 peptide were not recognized by 33 out of 36 CTL lines. The Tyr9 and Cys99 HLA-A2 molecules could bind the M1 55-73 peptide because at least one CTL line was found that could recognize each of these molecules that were exposed to the M1 peptide. CTL recognition patterns of variant M1 peptides presented by the Tyr9 molecule demonstrated that the amino acid at position 9 can be a critical determinant of the conformation of the peptide-A2 complex, and indicated that a particular peptide can bind in the HLA-A2 peptide binding groove in more than one conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies have shown that individual TCRs are able to effectively recognize multiple peptide/MHC complexes that have varying degrees of structural diversity. These TCR cross-reactivities have usually been demonstrated by using peptides that have different amino acid sequences. To further examine the extent to which TCRs can accommodate structurally diverse ligands, we analyzed human TCR cross-reactivity to eight structurally distinct haptens that are coupled to the HLA-A2-binding Tax peptide with a lysine substitution at position 5 (Tax-5K, LLFG[K-hapten]PVYV). The results demonstrate that 71% percent of the haptenated-peptide-induced CTL lines could cross-react on at least one other hapten. We compared the effects of HLA-A2 mutants with substitutions at known TCR contact sites for recognition by hapten-cross-reactive CTL. Recognition of the A2 mutants was remarkably similar whether they were presenting the immunizing or the cross-reactive peptide, indicating that similar amino acid contacts are made by the TCR during recognition of both complexes. Thus, hapten cross-reactivity is apparently accomplished without major adjustments to the interaction between the TCR and the surface of the HLA-A2 molecule. Collectively, these results suggest that TCRs possess the molecular flexibility to accommodate very structurally diverse ligands while retaining conserved interactions with the surface of the MHC molecule.

T cell recognition by peptide-specific αβ TCRs involves not only recognition of the peptide, but also recognition of multiple molecular features on the surface of the MHC molecule to which the peptide has been bound. We have previously shown that TCRs that are specific for five different peptides presented by HLA-A2 recognize similar molecular features on the surface of the α1 and α2 helices of the HLA-A2 molecule. We next asked whether these same molecular features of the HLA-A2 molecule would be recognized by hapten-specific HLA-A2-restricted TCRs, given that hapten-specific T cells frequently show reduced MHC dependence/restriction. The results show that a panel of CD8+ CTL that are specific for the hapten DNP bound to two different peptides presented by HLA-A2 do the following: 1) show stringent MHC restriction, and 2) are largely affected by the same mutations on the HLA-A2 molecule that affected recognition by peptide-specific CTL. A small subset of this panel of CD8+ CTL can recognize a mutant HLA-A2 molecule in the absence of hapten. These data suggest that TCR recognition of a divergent repertoire of ligands presented by HLA-A2 is largely dependent upon common structural elements in the central portion of the peptide-binding site.

The structures of αβ TCRs bound to complexes of class I MHC molecules and peptide show that the TCRs make multiple contacts with the α1 and α2 helixes of the MHC. Previously we have shown that the A6 TCR in complex with the HLA-A2/Tax peptide has 15 contact sites on HLA-A2. Single amino acid mutagenesis of these contact sites demonstrated that mutation of only three amino acids clustered on the α1 helix (R65, K66, A69) disrupted recognition by the A6 TCR. In the present study we have asked whether TCRs that recognize four other peptides presented by HLA-A2 interact with the MHC in identical, similar, or different patterns as the A6 TCR. Mutants K66A and Q155A had the highest frequency of negative effects on lysis. A subset of peptide-specific CTL also selectively recognized mutants K66A or Q155A in the absence of exogenous cognate peptides, indicating that these mutations affected the presentation of endogenous peptide/HLA-A2 complexes. These findings suggest that most HLA-A2-restricted TCRs recognize surfaces on the HLA-A2/peptide complex that are dependent upon the side chains of K66 and Q155 in the central portion of the peptide binding groove. Crystallographic structures of several peptide/HLA-A2 structures have shown that the side chains of these critical amino acids that make contact with the A6 TCR also contact the bound peptide. Collectively, our results indicate that the generalized effects of changes at these critical amino acids are probably due to the fact that they can be directly contacted by TCRs as well as influence the binding and presentation of the bound peptides.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system characterized by lesions that are areas of blood–brain barrier breakdown, inflammation and myelin damage. To identify genes that contribute to lesion pathology, we have compared gene expression in MS lesions and in brains of mice with experimental allergic encephalomyelitis (EAE) with that in normal white matter. Gene expression was analyzed by cDNA microarrays consisting of 2798 human genes. One of the genes found to be upregulated in both MS lesions and EAE brains was 5-lipoxygenase (5-LO), a key enzyme in the biosynthesis of the proinflammatory leukotrienes. The presence of 5-LO in MS lesions was confirmed by immunohistochemistry and indicated that 5-LO was primarily contained within macrophages. Although these findings are not specific for MS, they identify a potentially important component of pro-inflammatory activity in the demyelinating process in MS and suggest a possible target for anti-inflammatory therapy in MS.

Structural studies have shown that class I major histocompatibility complex (MHC)-restricted peptide-specific T cell receptor (TCR)-alpha/betas make multiple contacts with the alpha1 and alpha2 helices of the MHC, but it is unclear which or how many of these interactions contribute to functional binding. We have addressed this question by performing single amino acid mutagenesis of the 15 TCR contact sites on the human histocompatibility leukocyte antigen (HLA)-A2 molecule recognized by the A6 TCR specific for the Tax peptide presented by HLA-A2. The results demonstrate that mutagenesis of only three amino acids (R65, K66, and A69) that are clustered on the alpha1 helix affected T cell recognition of the Tax/HLA-A2 complex. At least one of these three mutants affected T cell recognition by every member of a large panel of Tax/HLA-A2-specific T cell lines. Biacore measurements showed that these three HLA-A2 mutations also altered A6 TCR binding kinetics, reducing binding affinity. These results show that for Tax/HLA-A2-specific TCRs, there is a location on the central portion of the alpha1 helix that provides interactions crucial to their function with the MHC molecule.

The crystal structure of the human class I MHC molecule HLA-A2 complexed with of an octameric peptide, Tax8 (LFGYPVYV), from human T cell lymphotrophic virus-1 (HTLV-1) has been determined. This structure is compared with a newly refined, higher resolution (1.8 Å) structure of HLA-A2 complexed with the nonameric Tax9 peptide (LLFGYPVYV) with one more N-terminal residue. Despite the absence of a peptide residue (P1) bound in the conserved N-terminal peptide-binding pocket of the Tax8/HLA-A2 complex, the structures of the two complexes are essentially identical. Water molecules in the Tax8 complex replace the terminal amino group of the Tax9 peptide and mediate a network of hydrogen bonds among the secondary structural elements at that end of the peptide-binding groove. Thermal denaturation measurements indicate that the Tax8 complex is much less stable, ΔTm = 16°C, than the Tax9 complex, but both can sensitize target cells for lysis by some Tax-specific CTL from HTLV-1 infected individuals. The absence of a P1 peptide residue is thus not enough to prevent formation of a “closed conformation” of the peptide-binding site. TCR affinity measurements and cytotoxic T cell assays indicate that the Tax8/HLA-A2 complex does not functionally cross-react with the A6-TCR-bearing T cell clone specific for Tax9/HLA-A2 complexes.

Down-regulation of the initial burst of viremia during primary HIV infection is thought to be mediated predominantly by HIV-specific cytotoxic T lymphocytes, and the appearance of this response is associated with major perturbations of the T cell receptor repertoire. Changes in the T cell receptor repertoire of virus-specific cytotoxic T lymphocytes were analyzed in patients with primary infection to understand the failure of the cellular immune response to control viral spread and replication. This analysis demonstrated that a significant number of HIV-specific T cell clones involved in the primary immune response rapidly disappeared. The disappearance was not the result of mutations in the virus epitopes recognized by these clones. Evidence is provided that phenomena such as high-dose tolerance or clonal exhaustion might be involved in the disappearance of these monoclonally expanded HIV-specific cytotoxic T cell clones. These findings should provide insights into how HIV, and possibly other viruses, elude the host immune response during primary infection.

To identify genes that are expressed in MS pathogenesis, we have analyzed a normalized cDNA library made from mRNA obtained from CNS lesions of a patient with primary progressive MS. Complementary DNA clones obtained from this library were subjected to automated DNA sequencing to generate expressed sequence tags. Analysis of this MS cDNA library revealed the presence of 54 cDNAs that were associated with immune activation and indicated the presence of an ongoing inflammatory response with evidence of both cell-mediated and humoral immune responses. The surprising finding was that 16 of the cDNAs encoded autoantigens associated with seven other autoimmune disorders, while only three of these 16 autoantigen cDNAs were present in a similarly constructed adult brain library. Such aberrant autoantigen expression could provide a source of secondary autoimmune stimulation that could contribute to the ongoing inflammatory response in MS. In addition, two cDNAs were found that mapped to a known MS susceptibility locus (5p14-p12): one encoded an excitatory amino acid transporter and the other a human homologue of the Drosophila disabled gene. This approach to the molecular biology of MS pathogenesis may help to illuminate previously unappreciated aspects of this disease.

Genetic makeup thought to affect susceptibility to multiple sclerosis (MS) and current evidence suggests that multiple genes may be involved. We have mapped a potential susceptibility gene or genes in the germ-line T cell receptor (TcR) V beta region of multiple sclerosis (MS) patients. Six restriction fragment length polymorphisms (RFLPs) spanning approximately 600 kb of the TcR V beta region were used to define TcR haplotypes in 197 Caucasian controls and 83 Caucasian MS patients in the chronic progressive stage of the disease. The distribution of TcR subhaplotype frequencies was significantly different only in the approx. 175-kb region between RFLPs defined by V beta 8.1 and V beta 11. Stratification of the MS patients into HLA-DR2+ (n = 51) and HLA-DR2- (n = 32) populations demonstrated that the subhaplotype frequencies differed from the control population significantly only in the HLA-DR2+ (corrected P = 0.00007) and not in the HLA-Dr2- (corrected P = 0.46) population. Subhaplotypes which are rare in the normal population are overrepresented in the HLA-DR2+ MS patient population and confer a relative risk of 4.06. These results indicate the existence of an MS susceptibility gene within the TcR V beta region, and provide new evidence for gene complementation between a HLA class II gene and TcR V beta gene(s) in conferring susceptibility to MS.

Six pockets, designated A through F, which extend from the peptide binding site of class I HLA molecules, have been postulated to play an important role in determining peptide binding specificity. HLA-B27 mutant molecules with single amino acid substitutions at residues 9his-->phe, 24thr-->ser, 45glu-->thr, and 67cys-->ala in pocket B; 114his-->asn in pocket D; and 116asp-->phe in pocket F have been generated and characterized for their capacity to present an influenza A nucleoprotein peptide (NP 383-391) for cytotoxic T lymphocyte recognition. We report here that substitutions in residues 45, 67, and 116 affect presentation of NP 383-391 when peptide is processed and loaded during viral infection. Using 125I-labeled NP peptide, we demonstrate that substitutions in residues 67 and 116 alter the stability of NP-HLA-B27 complexes. A substitution at position 9 of the NP peptide complements the mutation introduced at residue 116, suggesting that the NP peptide binds with its carboxy terminal amino acid in pocket F. These findings indicate that polymorphic residues within pockets B and F of HLA-B27 play a crucial role in peptide binding and stability of peptide-MHC class I complexes. Furthermore, our results suggest that substitutions at allele-specific residues within pockets B and F alter the stability of NP-HLA-B27 complexes resulting in the diminution or abrogation of NP presentation during viral infection.

To assess whether similar or dissimilar molecular features of class I human lymphocyte antigen (HLA) molecules determine the presentation of structurally diverse peptides, we have examined the influence of different pockets within the HLA-A2.1 molecule on the presentation of three different viral peptides. The influenza virus M1 58-66, HTLV-I Tax peptide 12-19, and HCMV gB 619-628 are minimal peptides that induce HLA-A2.1-restricted non-cross-reactive CTL responses. The influence of distinct structural features of HLA-A2.1 on peptide presentation was analyzed using a panel of 14 HLA-A2 mutants each with single amino acid substitutions in one of six pockets that are located in the peptide binding site. Ten of the 14 mutants showed concordant effects on the presentation of all three peptides to their peptide-specific CTL lines. Four of the mutants had a negative effect on the presentation of only one or two of these viral peptides. These findings indicate that common structural features in HLA-A2 determine the binding and conformation of different peptides, and help to provide a plausible explanation for how diverse peptides bind to HLA-A2.

Peripheral blood leukocytes (PBL) from 18 homosexual men who did not have acquired immunodeficiency syndrome (AIDS) and from 9 heterosexual men were repetitively tested for their ability to generate HLA self-restricted cytotoxic T lymphocyte responses to influenza virus (flu-self) over a 2-yr period. The sera of the same donors were tested for antibodies to human T lymphotropic virus-III (HTLV-III). Six of the homosexual and none of the heterosexual donors consistently generated weak cytotoxic T lymphocyte responses to flu-self. Seven of the homosexual and none of the heterosexual donors were seropositive for antibodies to HTLV-III. No obvious correlation was detected between weak flu-self cytotoxic T lymphocyte responses and antibodies to HTLV-III. However, one homosexual donor generated no detectable cytotoxic T lymphocyte activity to flu-self, although he was a strong responder to HLA-alloantigens. This donor had an OKT4:OKT8 ratio of 0.4 and was seropositive for HTLV-III antigens; HTLV-III virus was identified in his PBL; and he developed AIDS during the course of this study. A second donor with lymphadenopathy and who was seropositive for HTLV-III antigens exhibited marginal cytotoxic T lymphocyte activity to flu-self which he subsequently lost. PBL from two patients, one with Kaposi's sarcoma and one with generalized lymphadenopathy, were also tested for cytotoxic T lymphocyte responses to flu-self and to alloantigens. Both donors failed to generate cytotoxic T lymphocyte to flu-self, but generated strong cytotoxic T lymphocyte responses to alloantigens. The selective loss of an HLA-restricted cytotoxic T lymphocyte response without loss of HLA alloantigenic cytotoxic T lymphocyte activity may be an important functional immunologic characteristic in the development of AIDS.

A recently described HLA gene, SB, which maps between GLO and HLA-DR, codes for Ia-like molecules that are similar to but distinct from HLA-DR molecules. Cytotoxic T lymphocytes (CTL) specific for SB1, SB2, SB3, and SB4 were compared with HLA-A2-specific CTL with respect to their surface expression of the T cell differentiation antigens OKT3, OKT4, and OKT8. All CTL activity was eliminated by treatment with OKT3 and C'. The SB-specific cytotoxicity was eliminated by OKT4 plus C' but not by OKT8 plus C'. In contrast, HLA-A2-specific killing was completely susceptible to treatment with OKT8 plus C' but not with OKT4 plus C'. Cytotoxicity was analyzed in the presence of OKT8 and a series of monoclonal antibodies (OKT4A, 4B, 4C, and 4D) that react with distinct epitopes on the OKT4 molecule. SB1-, SB3-, and SB4-specific CTL were partially inhibited by OKT4A and 4B (45-75%), whereas HLA-A2-specific CTL were partially inhibited by OKT8 (48-63%) but not by OKT4. SB2-specific CTL were not inhibited (less than 26%) by OKT8 or by any of the OKT4-related antibodies. These results suggest that the OKT4 marker may be expressed on most T cells that recognize allogeneic Ia or self Ia plus foreign antigens; OKT4+ cells do not appear to be functionally homogeneous in that they can act both as helper/inducer and cytotoxic cells. Models are proposed for the functional involvement of the OKT4 molecule in T cell-Ia antigen interactions.

The CD4 molecule is currently the object of intense interest and investigation both because of its role in normal T-cell function, and because of its role in HIV infection. Our studies of CD4 expression and function on class II-specific T cells indicate that: 1) CD4 expression is generally a marker of T cells that are committed to class II antigen recognition, even when they have cytotoxic function; 2) CD4 is typically involved in the proliferative and cytotoxic function of such cells, which we hypothesized to be due to interaction with a nonpolymorphic determinant on class II; 3) the importance of CD4 to the recognition is most prominent in T-cell interactions with low "avidity"; and 4) although CD4 may play a role in adhesion per se, the majority of its function seems to be in a post-adhesion phase of cytotoxic T-cell recognition.

The self-specificity of human influenza virus-immune cytotoxic T cells has been analyzed in order to identify the relationship between the self-determinants which they recognize and the serologically defined HLA-A and -B antigenic determinants. Virus-immune T cells were generated in vitro by culture of normal adult peripheral blood lymphocytes with A/HK influenza virus. Virus-immune effectors from HLA-A2 positive donors were tested on panels of virus-infected target cells from donors who were either HLA-mismatched or matched only for the HLA-A2 specificity. Virus-immune T cells from 11/11 A2-positive donors lysed all 2A-matched virus-infected target cells (and no HLA-mismatched targets), except that each of these effector cell populations consistently failed to lyse the virus-infected target cells from ove A2-positive donor (designated M7). Although the A2 antigen of donor M7 could also be distinguished from the A2 antigen of other donors by alloimmune cytotoxic T cells, no differences in the A2 antigen of donor M7 could be defined by extensive serological analyses. Results of iscelectric focusing of A2 molecules from three individuals plus M7 demonstrated that the M7 A2 heavy polypeptide chain is structurally distinct. These results indicate that: 1) there is a strong but incomplete associatione between a self antigen recognized by virus-immune T cells and the serologically defined HLA-A2 specificity; and 2) there may be at least two structurally and functionally distinct epitopes on the same A2 molecule: one is the serologically defined HLA-A2 antigenic determinant; the other is the self determinant recognized by T cells on HLA-A2 molecules.

We have investigated elements of the genetic control of human in vitro cytotoxic T-cell responses to influenza virus-infected autologous cells by studies of a large family. The pattern of virus-immune cytotoxicity among siblings demonstrated T-cell recognition of influenza virus predominantly (greater than 90%) in association with determinants which are coded by genes linked to HLA (P less than 0.0002). Many family members consistently generated cytotoxic activity against influenza predominantly in association with antigens coded by genes of only one of their HLA haplotypes. Such haplotype preferences were consistent among HLA-identical siblings, indicating that the specificity of the T-cell response to influenza virus in association with HLA-A and -B antigens is controlled by genes linked to HLA.

The present report attempts to determine if there are distinct epitopes on the T8 molecule that are involved in class I-restricted cytotoxic T lymphocyte (CTL) function. A panel of 9 monoclonal antibodies (OKT8A,B,C,D,E,F,G,H,I, and OKT5) was produced and all antibodies were shown to bind to the T8 molecule. This panel of antibodies was employed to characterize the distribution of distinct epitopes on the T8 molecule and to block the activity of class I-specific influenza virus-immune and allo-immune CTL effector function. Significant differences in the ability of the anti-T8 antibodies to block CTL function were observed: OKT8C and T8F blocked best (49 and 55% respectively); OKT8A,E,G,H,I, and OKT5 blocked less well (24–31%); and OKT8B blocked marginally (11%). There was no correlation between the capacity of the antibodies to block CTL function and their heavy chain isotype. Competitive binding of the different OKT8 antibodies to the cell surface and differential trypsin sensitivity of the epitopes recognized by the antibodies indicated that OKT8C and T8F were located on topographically distinct regions of the T8 molecule. These results indicate that specific epitopes on the T8 molecule are involved in CTL function, and that there could be more than one functional site on the molecule.

The human major histocompatibility complex (HLA) has been demonstrated to play two roles in the generation and expression of cytotoxic T-lymphocyte responses to virus-infected cells: (1) cytotoxic T cells can only recognize viral antigens in conjunction with antigens encoded by HLA-A and -B genes; and (2) HLA-linked genes may control the capacity to generate T-cell responses to a given virus or to virus in conjunction with particular self HLA-A and -B antigens. Analysis of T-cell responses generated in vivo to Epstein-Barr virus suggests that human T cells may recognize virus in conjunction with antigens other than the class I HLA polymorphic specificities.

Secondary effector T-cell populations generated by cross-priming with heterologous influenza A viruses operate only in H-2K or H-2D compatible situations, when assayed on SV40-transformed target cells infected with a range of influenza A viruses. The H2-Kb allele is associated with a total failure in the generation of influenza-immune cytotoxic T cells, though this is not seen for the primary response to vaccinia virus. In both influenza and vaccinia development of effector T cells operating at H-2Db is greatly depressed in B10.A(2R) (kkkddb) and B10.A(4R) (kkbbbb), but not in B10 (bbbbbb), mice. However, there is no defect in viral antigen expression at either H-2Kk or H-2Db in B10.A(2R) target cells. This apparently reflects some inadequacy in the stimulator environment, as (A/J X B6) F1 T cells can be induced to respond at H-2Db when exposed to vaccinia virus in an irradiated B6 but not in a B10.A(4R) recipient. The present report, together with the accompanying paper by Zinkernagel and colleagues, records the first rigorous demonstration of both a nonresponder situation and a probable Ir-gene effect for conventional infectious viruses. Possible implications for the evolution of H-2 polymorphism and mechanisms of Ir gene function are discussed.

The specificity of in vitro induced human influenza-immune cytotoxic effector cells was analyzed with respect to recognition of HLA-A and -B-linked gene products. The influenza-immune cytotoxic activity observed on panels of virus-infected targets demonstrated that virus-immune effectors preferentially lyse targets with which they share HLA-A or -B specificities. Virus-immune effectors from certain donors recognized virus in conjunction with some, but not all, of their self HLA-A and -B antigens. Among donors who share a given HLA antigen (such as A2 or B7), there are differences in the ability of their virus-immune T cells to recognize the shared antigen. Virus-infected target cells from HLA-A2 or -B7 “nonresponder” donors could be lysed by virus-immune T cells obtained from other donors who shared only the HLA-A2 or -B7 antigen with these target cells. These observations suggest that the absence of cytotoxic T cell responses by some donors to influenza virus in conjunction with HLA-A2 or -B7 is not due to control by the structural genes that code for these HLA antigens, but rather may result from control by regulatory genes that act at the level of the responder and/or stimulator cell. The results are discussed in the context of Ir gene regulation of human T cell responses.

To determine whether healthy homosexual men are immunologically impaired, peripheral blood leukocytes (PBL) from 20 male homosexuals were compared prospectively with PBL from 14 age-matched male heterosexual donors with respect to: (a) the capacity of their PBL to generate functional T cell immune responses in vitro; and (b) the content of total T cells and T cell subsets in their peripheral blood. The homosexual donors studied indicated moderate sexual life styles in that all but one of the donors had less than five current sexual partners. The percentages of OKT3+, OKT4+, and OKT8+ T cells were similar to those of heterosexual controls. T cell function was assessed by measuring cytotoxic T cell responses to influenza virus and to allogeneic cells. Approximately one-third of the homosexual donors consistently exhibited weak cytotoxic T lymphocyte (CTL) responses to influenza virus, whereas all of the heterosexual donors generated strong CTL responses to influenza. There was no correlation between the strength of CTL responsiveness to influenza virus and the strength of CTL responses to allogeneic cells. These results suggest that the influenza-specific CTL response may be a sensitive indicator of immunologic defects in asymptomatic homosexuals. If acquired immune deficiency syndrome results from an infectious agent, it remains to be seen if such immunosuppression predisposes to the infection, or if it reflects early consequences of infection.

The immune reactivity to tumor cells within a progressively growing tumor mass in the syngeneic host has been analyzed by studying the cell-mediated cytolytic response of DBA/2 mice to the ascitic mastocytoma P815Y. Peritoneal cells from P815Y tumor-bearing hosts were fractionated by velocity sedimentation at unit gravity. Cell-mediated cytotoxicity of fractionated and unfractionated cells was measured by 51Cr-release from tumor target cells. The cell separation procedure revealed significant levels of specific cell-mediated cytotoxicity to P815Y within peritoneal cell populations at 8-16 days after tumor cell inoculation. Tumor cells purified from the peritoneal cell populations of mice injected with 10(3) tumor cells 10 days previously were as susceptible to syngeneic and allogeneic cell-mediated cytotoxicity as P815Y grown in vitro. However, tumor cells obtained from mice 16 days after tumor inoculation were resistant to cytolysis by syngeneic, but not allogeneic, effector cells. In addition, day 16 tumor cells did not inhibit syngeneic cell-mediated cytotoxicity against P815Y grown in vitro. Immunoglobulin was not detected on day 16 tumor cells and no circulating antibody to P815Y was found in the ascitic fluid of day 16 tumor-bearing mice. These results indicate that tumor cells may escape immune attack by loss of expression of cell surface tumor-associated antigens in the absence of circulating antibody against tumor.

By using a panel of HLA-D-defined subtypes of HLA-DR2 HCL with known beta chain structural variabilities, we have demonstrated that HLA-DR2, OKT4+ cytotoxic T lymphocyte (CTL) clones specific for measles virus are apparently restricted to a distinct DR beta chain. The presence of this DR beta 2 molecule correlated precisely with the susceptibility of measles virus-infected HLA-DR2 HCL to lysis by these CTL clones. These studies demonstrate that delineation of HLA-DR2 into various subgroups can have a functional significance that parallels the structural differences within the HLA-D region. These results are discussed in the context of the possible association of HLA class II-restricted, measles virus-specific CTL and multiple sclerosis.

Cytotoxic thymus-derived lymphocytes generated after interaction with trinitrophenyl (TNP)-substituted or virus-infected cells only lyse H-2 compatible target cells modified with the component used to immunize (TNP or virus). Prior saturation of TNP-reactive sites inhibits neither the infectivity of influenza A viruses, nor the capacity of infected cells to develop antigenic changes recognized by influenza-immune T cells. The two antigens are distinct entities on the cell membrane and do not obviously compete to form interactions with H-2 molecules.

Murine splenocytes immune to influenza virus-activated human T-cells were fused with SP2/0 cells, selected in chemically defined HAT media, and subcloned to yield a monoclonal antibody (MAb) termed 7G7/B6. 7G7/B6 binds to lectin- and antigen-activated T-cells, but not resting T-cells or B-lymphoblastoid lines from the same donor. 7G7/B6 immunoprecipitates a 50-55 kD band from cell surface iodinated PHA-activated T-cells or the T-cell leukemia line HUT 102B2, as shown on SDS-PAGE. Cross-clearing studies demonstrate that 7G7/B6 binds the same cell surface molecule(s) as anti-Tac, a MAb which has been shown previously to recognize the human receptor for IL-2. 35S-methionine pulse chase experiments in HUT 102B2 cells reveal that 7G7/B6 binds to an early (less than 30 min) 35-37 kD and late (greater than 4 h) 50 kD protein. Sequential immunoprecipitations demonstrate that these are identical to the molecules identified by anti-Tac under similar conditions. However, only anti-Tac coprecipitates a higher molecular band at 110 kD. 7G7/B6 and anti-Tac do not competitively inhibit the binding of each other to PHA-activated T-cells. Functional studies reveal that in contrast to anti-Tac, 7G7/B6 has almost no inhibitory effect in vitro on IL-2-driven proliferation of IL-2-dependent T-cell lines, or alloimmune cytotoxic T-cell generation (however, once generated, these cytotoxic T-cells were both 7G7/B6 and anti-Tac positive). Finally, IL-2 does not inhibit the binding of 7G7/B6 to activated T-cells under conditions which result in up to 75% inhibition of anti-Tac binding. Therefore, 7G7/B6 is another MAb recognizing the human IL-2 receptor, but binding to an epitope distinct from that recognized by either IL-2 or anti-Tac.

In the present study cytotoxic T lymphocytes were generated in MLC of lymphocytes from two unrelated HLA-A, B, C-identical, B27-positive, but D/DR-different, individuals. These CTL were shown to detect subtypes of HLA-B27. CTL specific for influenza virus lysed infected target cells matched for HLA-B27 only when they shared the same subtype. This indicates that the two subtypes of HLA-B27 detected by CTL function also as distinct elements in a self-restricted CTL response. Both subtypes were found among patients with ankylosing spondylitis.